Method of controlling telomere length

ABSTRACT

A method of controlling telomere length wherein the method comprises introducing into a cell a DNA encoding Mre11 protein or a DNA encoding a protein comprising Mre11 protein wherein a part of the nuclease domain, the C-terminal domain or the whole thereof is modified or deleted.

TECHNICAL FIELD

This invention relates to a method of controlling telomere lengthwherein physiological activity of endogenous Mre11 protein in aeukaryotic cell is modified, a telomere length controlling agentcomprising as an active component a substance which modifiesphysiological activity of endogenous Mre11 protein in a eukaryotic cell,and a gene therapeutic agent for telomere length-associated diseasescomprising as an active component a substance which modifiesphysiological activity of endogenous Mre11 protein in a eukaryotic cell.

BACKGROUND ART

Telomeres are functional constructs existing at the ends ofdouble-stranded DNA constituting filamentous chromosomes in a eukaryoticcell. It is known that this construct is generally composed of simplerepeats, and plays an important role in preventing chromosomalaberration due to chromosome fusion, pairing of homologous chromosomesduring myosis, control of gene expression, and determination of forms ofchromosomes in a nucleus.

In general, replication of a double-stranded DNA is carried out bysynthesizing a RNA primer by primase and at an end thereof byelongation-synthesis of a DNA chain by DNA polymerase. The RNA primer isremeved by 5′-3′ exonuclease activity of the DNA polymerase, and at thesame time the removed part is replaced with the DNA chain by the DNApolymerase. However, in the case of a filamentous chromosome, there isno replacement of the RNA primer at the 5′-most end of a nascent chainwith a DNA chain, and thus when replication is completed, the chromosomeis shortened by the size of the RNA primer. Therefore, every repeat ofcell division progressively shortens the chromosomes from its end in thedaughter cells, and finally the chromosomes become unstable, resultingin death of the cell. On the other hand, it has been shown that in animmortalized cell, telomerase which can elongate telomeres is highlyexpressed, thus telomere lengths can not be reduced. Consequently,intensive studies are made on regulators for cell life span oranticancer agents which involve the telomerase activity in theirmechanisms.

However, recently it has been shown that in yeasts and mice etc. evenwhen their telomerase genes are disrupted, it takes time for shorteningtheir teromeres [Blasco, M. A., et al.: Cell, 91:25-34 (1997); Rudolph,K. L., et al.: Cell, 96:701-12 (1999); Herrera, E., et al.: Embo J,18:2950-60(1999)], and that even a variant of a gene which is notdirectly related to telomerase activity, results in shortening orelongation of telomeres [Boulton, S. J., et al.: Embo J, 17:1819-28(1998); Furuse, M., et al.: Embo J, 17:6412-25(1998); Wilson, S. et al.:Nucleic Acids Res, 27:2655-61 (1999)]. Thus, it is pointed out thatthere are potentially mechanisms other than telomerase for maintainingtelomeres [Nakamura, T. M., et al.: Science, 282:493-6 (1998); Reddel,R. R., et al. A review. Biochemistry (Mosc), 62:1254-62 (1997)]. Inparticular, it has been reported that a Mre11-Rad50-Xrs2 complex whichis known to be engaged in repair of double-stranded DNA cleavage etc. isalso associated with controlling telomere length [Boulton, S. J., etal.: Embo J, 17: 1819-28 (1998); Furuse, M., et al.: Embo J,17:6412-25(1998); Le, S., et al.: Genetics, 152:143-52 (1999)]. AMre11-Rad50-Xrs2 (Nbsl) complex [Ajimura, M., et al.: Genetics,133:51-66 (1993); Johzuka, K., et al.: Genetics, 139:1521-32 (1995)] isa protein complex formed with Mre11 as a core which has a strong DNAbinding activity and functions as a double-stranded DNA exonuclease anda single-stranded endonuclease [Furuse, M., et al.: Embo J, 17:6412-25(1998); Paull, T. T., et al.: Mol Cell, 1:969-79 (1998); Usui, T., etal.: Cell, 95:705-16 (1998); Trujillo, K. M., et al.: J Biol Chem,273:21447-50 (1998)]. The complex functions as an essential enzyme toinitiation reaction for repair of DNA double-stranded cleavage orchromosomeal recombination. In budding yeast, Saccharomyces cerevisiae,or fission yeast, Schizosaccharomyces pombe, when even one of theproteins constituting this enzyme-complex is absent, notable shorteningof telomeres is observed [Boulton, S. J., et al.: Embo J,17:1819-28(1998)]. Homologs of all the constituent proteins of theMre11-Rad50-Xrs2(Nbs1) complex exist in humans [Dolganov, G. M., et al.:Mol Cell Biol, 16:4832-41 (1996); Petrini, J. H., et al.: Genomics,29:80-6 (1995)], and it is thought that they have a common role in alleukaryotes from yeast to human.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method ofcontrolling telomere length wherein the method comprises modifyingphysiological activity of endogenous Mre11 protein in a eukaryotic cell.

As a result of intensive studies based on the above subject, theinventors of the present invention have found that modifyingphysiological activity of endogenous Mre11 protein in a eukaryotic cellenables control of telomere length of the cell, thus accomplishing thepresent invention.

Namely, the present invention is a method of controlling telomere lengthwherein the method comprises modifying physiological activity ofendogenous Mre11 protein in a eukaryotic cell. Herein, the modificationof the physiological activities of the endogenous Mre11 protein iscarried out by introducing into a cell a DNA encoding foreign Mre11protein, or a DNA encoding a protein wherein the nuclease domain or theC-terminal domain of the foreign Mre11 protein is modified, in a statesuch that the DNA can be expressed.

Further, the present invention is a telomere length controlling agent ora gene therapeutic agent for telomere-associated diseases (e.g.melanoma, hepatoma, breast cancer, gastric cancer, brain tumor, and cellsenescent diseases) comprising as an active agent a substance whichmodifies physiological activity of endogenous Mre11 protein in aeukaryotic cell. Herein, the substance which modifies physiologicalactivity of endogenous Mre11 protein in the eukaryotic cell may includea DNA construct which comprises a DNA encoding foreign Mre11 protein, ora DNA encoding a protein wherein a nuclease domain or a C-terminaldomain of foreign Mre11 protein is modified, in a state such that theDNA can be expressed.

In the present invention, the following protein (a) or (b) is providedas foreign Mre11 protein:

-   (a) a protein comprising an amino acid sequence represented by SEQ    ID NO: 2 or 4; or-   (b) a protein comprising an amino acid sequence which is the amino    acid sequence represented by SEQ ID NO: 2 or 4 from, in, or to which    one or more amino acids are deleted, substituted or added, and    having physiological activity of Mre11 protein.

Further, in the present invention, the following DNA (c) or (d) isprovided as the DNA encoding foreign Mre11 protein:

-   (c) a DNA comprising a nucleotide sequence represented by SEQ ID NO:    1 or 3; or-   (d) a DNA which can hybridize with the DNA of (c) under a stringent    condition and encodes a protein having physiological activity of    Mre11 protein.

Furthermore, in the present invention, the following protein (e) or (f)is provided as a protein wherein a nuclease domain of the foreign Mre11protein is modified:

-   (e) a protein comprising an amino acid sequence represented by SEQ    ID NO: 6; or-   (f) a protein comprising an amino acid sequence which is the amino    acid sequence represented by SEQ ID NO: 6 from, in, or to which one    or more amino acids are deleted, substituted or added, and having    physiological activity (except nuclease activity) of Mre11 protein.

Moreover, in the present invention, the following DNA (g) or (h) isprovided as DNA encoding a protein wherein the nuclease domain of theforeign Mre11 protein is modified:

-   (g) a DNA comprising a nucleotide sequence represented by SEQ ID No:    5; or-   (h) a DNA which can hybridize with the DNA of (g) under a stringent    condition and encodes a protein having physiological activity    (except nuclease activity) of Mre11 protein.

Further, in the present invention, the following protein (i) or (j) isprovided as a protein wherein a C-terminal domain of the foreign Mre11protein is modified:

-   (i) a protein comprising an amino acid sequence represented by SEQ    ID NO: 8; or-   (j) a protein comprising an amino acid sequence which is the amino    acid sequence represented by SEQ ID NO: 8 from, in or to which one    or more amino acids are deleted, substituted or added, and having    physiological activity (except double-stranded DNA binding activity)    of Mre11 protein.

Still further, in the present invention, the following DNA (k) or (l) isprovided as DNA encoding a protein wherein the C-terminal domain of theforeign Mre11 protein is modified:

-   (k) a DNA comprising a nucleotide sequence represented by SEQ ID NO:    7; or-   (l) a DNA which can hybridize with the DNA of (k) and encodes a    protein having physiological activity (except double-stranded DNA    binding activity) of Mre11 protein.

The present invention will hereinafter be described in detail.

A method of controlling telomere length according to the presentinvention is based on modification of physiological activity ofendogenous Mre11 protein in a eukaryotic cell. Herein, the term“controlling telomere length” means shortening, retaining or elongatingtelomere length in cells. The term “endogenous Mre11 protein” means aprotein which natively exists in a cell and has physiological activityof Mre11 protein. The term “physiological activity of Mre11 protein”means at least one of the following activities: the ability of forming acomplex with Rad50 protein and Xrs2 (Nbs1) protein, double-stranded DNAbinding activity, double-stranded DNA exonuclease activity andsingle-stranded DNA endonuclease activity. The double-stranded DNAexonuclease activity and the single-stranded DNA endonuclease activityboth as a whole are referred to as the nuclease activity. Further,“modification” means to improve, reduce or delete, partially or whollyphysiological activity of the endogenous Mre11 protein. For example,shortening of telomeres can be carried out by introducing into a cell aDNA encoding a full length of foreign Mre11 protein or a DNA encoding aprotein wherein the nuclease domain of the foreign Mre11 protein ismodified, in a state such that the DNA can be expressed. Here, “a statesuch that the DNA can be expressed” means a state wherein the DNA iscomprised in a vector together with a DNA region concerning theexpression, e.g. a promoter so that a protein encoded by the DNA can beproduced in the cell into which the DNA has been introduced. Incontrast, the retaining or increasing of telomere length is carried outby introducing into a cell a DNA encoding a protein wherein theC-terminal domain of the foreign Mre11 protein is modified, in a statesuch that the DNA can be expressed. Here the term “foreign Mre11protein” means Mre11 protein derived from outside of a target cell, andthe term “modify” means that one or more amino acids is deleted,substituted or added from, in or to a functional domain.

1. Preparation of a DNA Encoding Foreign Mre11 Protein

(1) Sources of a DNA Encoding Foreign Mre11 Protein

The source of a DNA encoding foreign Mre11 protein which can be used forcontrolling telomere length is any organism-derived cell as long as ithas a DNA encoding Mre11 protein, without particular limitation.Examples thereof include cells derived from various eukaryotes e.g.human (Homo sapiens), a mouse (Mus musculus), a South African clawedfrog (Xenopus laevis), a fly (Drosophila melanogaster), Arabidopsisthaliana, Saccharomyces cerevisiae, Coprinus cinereus,Schizosaccharomyces pombe. Preferably a DNA encoding foreign Mre11protein to be used for controlling telomere length can be derived fromthe same species as that of a target cell for telomere length control.In regard to Schizosaccharomyces pombe, a protein corresponding to Mre11protein is called Rad32. For example, for controlling telomere length ofhuman cells, it is preferable to use a DNA encoding human Mre11 protein,and for controlling the telomere lengths of Saccharomyce cerevisiaecells, it is preferable to use a DNA encoding Mre11 protein fromSaccharomyce cerevisiae.

(2) Preparation of a DNA Encoding Foreign Mre11 Protein

A DNA encoding foreign Mre11 protein can be obtained by screening aclone including a DNA encoding Mre11 protein from a genome DNA libraryor a cDNA library which has been prepared from a cell derived fromorganisms described in the section (1) above, or by direct amplificationby PCR using a genome DNA or a cDNA as a template.

For example, for obtaining the DNA encoding Mre11 protein by screening acDNA library, initially mRNAs are prepared from cells of the organismsdescribed in section (1) above, by a method such as the guanidinethiocyanate method. Next, a synthetic DNA primer including an oligo-dTsequence is hybridized, then a single-stranded cDNA is synthesized by areverse transcriptase. Thereafter, using E coli DNA polymerase I, E coliDNA ligase and RNaseH, double-stranded cDNAs are synthesized by aconventional method. Next, after blunting cDNA ends by T4DNA polymerase,a DNA adaptor e.g. EcoRI adaptor, is added to both ends of the cDNAchains by T4DNA ligase. Then, the cDNA chains added by the DNA adaptorsare inserted into a restriction site of a commercially available λ phagevector (e.g. λZAP available from Stratagene) or a plasmid vector (e.g.pGEM2 available from PromegaBiotech) in accordance with a conventionalmethod. Thus, a group of recombinant λ phage DNAs or recombinant plasmidDNAs can be obtained.

Subsequently, in regard to the group of therecombinant λ phage DNAs, invitro packaging is performed using a commercially available in vitropackaging kit (e.g. Gigapack Gold available from PromegaBiotech),thereby producing an λ phage particles containing the recombinant λphage DNAs. The obtained λ phage particles are transfected into hostcells, e.g. E. coli, in accordance with a conventional method [Maniatis,T., et al.: Molecular Cloning. A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, 1989]. The obtained transfectants are allowedto proliferate, thereby a phage cDNA library can be obtained.

On the other hand, in regard to a group of the recombinant plasmid DNAs,for example, plasmid DNAs are transformed into host cells, e.g. E. coliin accordance with a conventional method. The obtained transformant isallowed to proliferate, thereby a plasmid cDNA library can be obtained.

Next, the above transformants are allowed to proliferate and transferredonto a nylon membrane or a nitrocellulose membrane , e.g. Gene ScreeningPlus available from DuPont, and proteins are removed under a alkalinecondition. The λ phage DNAs or plasmid DNAs on the membrane arehybridized with a radioactive-labeled probe derived from a partialfragment of a DNA encoding Mre11 protein, then positive clones areselected. Herein, the probe to be used can be prepared by PCR usingoligonucleotide primers designed based on a nucleotide sequence of a DNAencoding a partial amino acid sequence of Mre11 protein. For example,for PCR-preparation of a probe to be used for screening clones includinga DNA encoding yeast Mre11 protein, 5′-gtgccattattatttcagaa-3′ (SEQ IDNO: 9) and 5′-gggatcaagtacaactattttc-3′ (SEQ ID NO: 10) may be used as aprimer. Then, the obtained positive clones are cultured, and phage DNAsor plasmid DNAs are prepared from the culture.

In addition, for preparing a DNA encoding Mre11 protein by directPCR-amplification, initially genome DNA or cDNA are prepared from cellsof the organisms described in section (1) above. Then, using theobtained genome DNA or cDNA as templates, a DNA encoding an amino acidsequence at N-terminal side of the Mre11 protein and a DNA encoding anamino acid sequence at C-terminal side thereof are used as primers, PCRcan be performed. Next, a PCR-amplified fragment is ligated to asuitable vector. Examples of primers to be used for amplifying DNAencoding yeast Mre11 protein include 5′-atggactatcctgatccaga-3′ (SEQ IDNO: 11) and 5′-gggatcaagtacaactattttc-3′ (SEQ ID NO: 12). In the case ofobtaining the DNA encoding the Mre11 protein with directPCR-amplification, pfu polymerase is preferably used as a DNA sythetasedue to its infrequent misreading.

The DNA obtained according to the above method is verified to be a DNAencoding the Mre11 protein through determination of a nucleotidesequence by any of known methods, e.g. the dideoxy method. Determinationof a nucleotide sequence can usually be carried out by use of anautomatic nucleotide sequencer (e.g. 373A DNA sequencer manufactured byPERKIN-ELMER).

In SEQ ID NOS: 1 and 3, nucleotide sequences of the DNAs encoding theMre11 proteins derived from Saccharomyces cerevisiae and human,respectively, and in SEQ ID NOS: 2 and 4, the amino acid sequences ofthe proteins thereof are exemplified. However, as long as the proteinhas physiological activity of Mre11 protein, there may occur variationssuch as deletions, substitutions, and additions of one or more aminoacids relative to these amino acid sequences.

For example, one or more, preferably about 20 to 30, more preferably 10to 20 amino acids may be deleted from the amino acid sequencerepresented by SEQ ID NOS: 2 or 4. Or one or more, preferably about 20to 30, more preferably 10 to 20 amino acids may be added to the aminoacid sequence represented by SEQ ID NOS: 2 or 4. Alternatively, one ormore, preferably about 20 to 30, more preferably 10 to 20 amino acidsmay be substituted by other amino acid(s) in the amino acid sequencerepresented by SEQ ID NOS: 2 or 4.

Further, a DNA which can hybridize with the above-mentioned gene under astringent condition, as long as it encodes a protein havingphysiological activity of Mre11 protein, can be used in the presentinvention. The term “a stringent condition” means that, for example, thesodium concentration is from 33 to 70 mM, preferably 50 to 66 mM and thetemperature is from 40 to 70° C., preferably 55 to 68° C.

The physiological activity of Mre11 protein means, as mentioned above,double-stranded DNA binding activity, double-stranded DNA exonucleaseactivity and single-stranded DNA endonuclease activity, and each ofthese activities can be determined in accordance with a method of Furuseet al. [Furuse et al.: EMBO J 17:6412-25 (1998)]

2. Introduction of Variations into a DNA Encoding Foreign Mre11 Protein

When a DNA encoding a variant of foreign Mre11 protein with modificationin its nuclease domain, is introduced into a cell in a state such thatthe DNA can be expressed, the telomeres are shortened more than in thecase where a DNA encoding a wild-type Mre11 protein is introduced.However, when a DNA encoding a variant of foreign Mre11 protein withmodification in its C-terminal domain, is introduced in a state suchthat the DNA can be expressed, the telomere length is retained orincreased, differing from the case where a DNA encoding a wild-typeMre11 protein is introduced. Here the term “nuclease domain” means, inan amino acid sequence constituting the Mre11 protein, an amino acidsequence responsible for double-stranded DNA exonuclease activity andsingle-stranded DNA endonuclease activity of the protein. In particular,for example with respect to yeast Mre11 protein, the nuclease domaincorresponds to a region from 225th to 450th amino acid numbered from theN-terminal. The term “C-terminal domain” means, in an amino acidsequence constituting the Mre11 protein, an amino acid sequence atcarboxyl terminal region responsible for double-stranded DNA bindingactivity of the protein. In particular, for example with respect toyeast Mre11 protein, the C-terminal domain corresponds to a region from600th amino acid onwards numbered from the N-terminal.

A DNA encoding a variant of Mre11 protein can be prepared as follows.For example, for preparing a DNA encoding a protein having specific oneor more amino acid in the nuclease domain of the Mre11 proteinsubstituted by different amino acid(s), there can be employed forexample a site-directed mutagenesis method based on PCR [Saigo Kaoru etal.: “BUNSHISEIBUTSU-GAKU JIKKEN PROTOCOL I (Experiment Protocol I forMolecular Biology), p. 263 -270, Maruzen, 1997], which is known in thetechnical field of the present invention.

3. Introduction of a DNA Encoding Mre11 Protein or a Variant of Mre11Protein into a Cell

A DNA encoding Mre11 protein or a variant of Mre11 protein can beintroduced into a cell as mentioned below. That is, after preparing arecombinant vector which includes a DNA encoding the Mre11 protein orvariant tehreof obtained in section 1 or 2 above, the obtainedrecombinant vector is introduced into a host cell.

(1) Preparation of a Recombinant Vector

The recombinant vector can be obtained by ligating (inserting) to asuitable vector a DNA encoding the Mre11 protein or the variant of Mre11protein. Herein, the vector to be used is not particularly limited, aslong as it is replicable in the host cell. Examples thereof include aplasmid vector and a virus vector.

A plasmid vector for a yeast host cell may be pMAC561aur, YEp13, YEp24,YCp50 etc., and a virus vector for an animal host cell may be aretrovirus, a vaccinia virus and the like.

For ligating the DNA encoding the Mre11 protein or the variant of Mre11protein to the vector, there may be employed a method wherein initiallya DNA fragment including the above DNA is cleaved by (an) appropriaterestriction enzyme(s), and then it is ligated to (a) restriction site(s)or a multi cloning site in the vector DNA.

It is necessary that the DNA encoding the Mre11 protein or variant ofMre11 protein should be incorporated into the vector in a state suchthey can be expressed in the host cell. Thus, in addition to a promoterand the DNA encoding the Mre11 protein or the variant of Mre11 protein,a cis element such as an enhancer, a splicing signal, a poly A additionsignal and a selective marker can be ligated as desired. Here, examplesof the selective marker include a dihydrofolate reductase gene, ablasticidin resistant gene, an aureobasidin resistant gene, and a G418resistant gene.

Incidentally, a recombinant vector pMACMre11D16A including a DNAencoding Mre11 protein having the asparatic acid substituted by alanineat the 16th amino acid position from an N-terminal of the Mre11 protein,and a recombinant vector pMACMre11 ΔC49 including a DNA encoding Mre11protein having a C-terminal domain deleted each have been introducedinto a E coli DH5α strain (ERKNMRE11D 16AYOPAUR and ERKNMRE11DC49YOPAUR)and deposited on Dec. 22, 1999, at the National Institute of Bioscienceand Human-Technology, the National Institute of Advance IndustrialScience and Technology, under the Ministry of Economy, Trade andIndustry, (Higashi 1-1-3, Tsukuba, Ibaraki, Japan) under the accessionNos. FERM BP-7421 and FERM BP-7422.

(2) Preparation of a Transformant Cell

Preparation of a transformant cell can be carried out by introducing therecombinant vector into a host such that a gene of interest can beexpressed. Herein, the host may be, without particularly limitation, anyeukaryotic cell as long as it has a filamentous chromosome includingtelomeres. Examples thereof include yeasts such as Saccharomycescerevisiae and Schizosaccharomyces pombe, animal cells such as COS cellsand CHO cells, or an insect cells such as Sf9, Sf21.

When using a yeast as a host, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris etc. may be used. In thiscase, any promoter may be used, without particularly limitation, thatcan be expressed in the yeast. Examples thereof include a gal1 promoter,a gal10 promoter, a heat shock protein promoter, a MFα1 promoter, a PH05promoter, a PGK promoter, a GAP promoter, an ADH promoter, an AOX1promoter. The method of introducing the recombinant vector into theyeast may be any of methods by which a DNA can be introduced into theyeast, without particular limitation, for example, electroporation[Becker, D. M. et al.: Methods. Enzymol., 194: 182(1990)], spheroplastmethod [Hinnen, A. et al.: Proc. Natl. Acad. Sci., USA, 75:1929(1978)],and lithium acetate method [Itoh, H.: J. Bacteriol., 153: 163 (1983)].

When using an animal cell as a host, a monkey COS-7 cell, Vero, aChinese hamster ovary cell (CHO cell), a mouse L cell, a rat GH3, or ahuman FL cell can be used. As a promoter, an SRα promoter, an SV40promoter, an LTR promoter, a CMV promoter or the like may be used, andan human cytomegalovirus early gene promoter etc. may be used as well.Exemplary methods for introducing the recombinant vector into the animalcell include electroporation, the lithium acetate method and thelipofection method.

When using an insect cell as a host, an Sf9 cell, an Sf21 cell or thelike may be used. Exemplary methods for introducing the recombinantvector into the insect cell include the calcium phosphate method, thelipofection method, electroporation and the like.

4. Analysis of Telomere Length

Telomere length can be analyzed by Southern hybridization using a probewhich specifically hybridizes to a telomere sequence. For example, foranalyzing the telomere length in the yeast cells, initially the genomeDNAs are extracted from control yeast cells and test yeast cells. Next,the obtained genome DNAs are digested by (an) appropriate restrictionenzyme(s) (e.g. XhoI), and then the DNA fragments are isolated byagarose gel electrophoresis. Thereafter, they are transcribed onto anylon membrane under an alkaline condition, and they are hybridized to alabeled-probe which specifically hybridizes with a telomere sequenceunder an appropriate condition. Herein, the probe may be anoligonucleotide comprising a sequence of 5′-gtgtgtgtgtgtgtgtgtgt-3′ (SEQID NO: 13). After washing the membrane, they are visualized withautoradiography or imaging plate (e.g. BAS2000 manufactured by FujiPhoto Film Co., Ltd.). Comparison between lanes of the control yeastcells and lanes of the test yeast cells showed clearly the difference intheir telomere length.

5. Application as an Agent for Controlling Telomere Length and a GeneTherapeutic Agent for Telomere Length-Related Diseases

By modifying physiological activity of the endogenous Mre11 protein in aeukaryotic cell, it is possible to control telomere length in the cell.Therefore, an agent comprising as an active component a substance whichmodifies physiological activity of endogenous Mre11 protein in aeukaryotic cell, is valuable as a control agent for telomere length anda gene therapeutic agent. Examples of the substance which modifiesphysiological activity of endogenous Mre11 protein in a eukaryotic cell,include a DNA construct comprising a DNA encoding Mre11 protein or a DNAencoding a protein wherein the nuclease domain or the C-terminal domainof the Mre11 protein is modified in a state such that the DNA can beexpressed.

(1) An Agent for Controlling Telomere Length

A DNA construct comprising a DNA encoding the Mre11 protein a proteinwherein the nuclease domain or the C-terminal domain of the Mre11protein is modified in a state such that the DNA can be expressed, isuseful as a agent for controlling the telomere length (e.g. a reagentfor telomere length control). That is, it is possible to controltelomere length in any cell derived from any organism having achromosome containing telomeres. To facilitate shortening of telomeres,a DNA construct comprising a DNA encoding the Mre11 protein or a proteinhaving modified a nuclease domain of the nuclease domain of the Mre11protein in a state such that either DNA can be expressed, is introducedinto the cell. In contrast, to retain or increase telomere length, a DNAconstruct comprising a DNA encoding a protein wherein the C-terminaldomain of the Mre11 protein is modified in a state such that the DNA canbe expressed, is introduced into the cell.

The method of introducing a gene into a yeast cell, may be any methodwithout particular limitation as long as it is a method of introducing aDNA into a yeast, and examples thereof include electroporation [Becker,D. M. et al.: Methods. Enzymol., 194: 182(1990)], spheroplast method[Hinnen, A. et al.: Proc. Natl. Acad. Sci., USA, 75:1929(1978)], andlithium acetate method [Itoh, H.: J. Bacteriol., 153: 163 (1983)].Exemplary methods of introducing a gene into an animal cell includeelectroporation, calcium phosphate method, and lipofection method.

(2) Gene Therapeutic Agent for Telomere Length-Associated Diseases

The term “telomere length-associated diseases” means any disease whichrelates to telomere length for the onset thereof. Examples thereofinclude malignant tumors (e.g. melanoma, hepatoma, breast cancer,gastric cancer, brain tumor) in which shortening of telomeres in theirchromosomes does not occur because of their increased telomeraseactivity, and senile diseases wherein shortening of telomeres isenhanced compared with a normal cell. When a DNA construct comprising aDNA encoding the Mre11 protein or a DNA encoding a protein wherein thenuclease domain or the C-terminal domain of the Mre11 protein. ismodified in a state such that the DNA can be expressed, is used as thegene therapeutic agent for telomere length-associated diseases, the DNAconstruct may be directly administered to a subject by methods such asinjection, gene gun etc. Examples of vector which can be used for theDNA construct, include an adenovirus vector, an adeno-associated virusvector, a herpes virus vector, a vaccinia virus vector, a retrovirusvector etc. These virus vectors enable the DNA to be more efficientlyintroduced into the cell. Further, a method may be employed whichcomprises introducing the DNA construct into a phospholipid vesicle suchas liposome and administering the liposome to a subject. In this method,since the liposome is a closed vesicle including a bio-degradablematerial, mixing the liposome with the gene of the present inventionallows the gene hold in the lipid bilayer or the internal aqueous layerof the liposome (liposome-gene complex). Next, by culturing a cell inthe presence of the complexes the gene in the complex is incorporatedinto the cell (lipofection method). Then, the obtained cell may beadministered to a subject according to administration methods describedbelow.

As an administration form of the gene therapeutic agent of the presentinvention, there may be employed local administration into anepithelical tissue (epidermis) or any of various organs or tissues etc.in addition to general systemic administration e.g. intravenous orintraarterial administration etc.. Moreover, an administration form incombination with catheter technique, surgical operation etc. can beemployed.

The dose of the gene therapeutic agent of the present invention isvaried, depending on the age, the sex, the condition of the patient, theroute or the frequency of administration, or the formulation type, butusually the range from 0.1 to 100 mg/body/day of the gene of the presentinvention is appropriate for adults.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of a structure of a recombinant vector.

FIG. 2 is a schematic view of an arm of an yeast chromosome.

FIG. 3 is a photograph of gel electropolation showing telomere length ofa yeast cell introduced with a recombinant vector .

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will hereinafter be described in detail withreference to Examples, but the scope of the present invention is notlimited thereto.

EXAMPLE 1

Preparation of a DNA Recombinant Vector Encoding a Yeast Mre11 Proteinand a Variant Yeast Mre11 Protein

(1) Preparation of a DNA Encoding Mre11 Protein

E. coli cells containing a genomic DNA library of yeast DBY939 strainconstructed by using a YEp24 yeast—E. coli shuttle vector (constructedby M. Carlson) were plated onto LB agar medium containing ampicillin,and the obtained colonies were transfer to a nylon membrane, then theDNA derived from the colonies onto the membrane in accordance with aconventional method. The polymerase-chain-reaction (PCR) was performedwith oligonucleotides as primers, each of which has a sequencecorresponding to the DNA sequence encoding the N-terminal region and theC-terminal region of the MRE11 protein respectively, and the genome DNAof wild-type blast yeast strain ORD149 as a template. The obtained DNAfragment was cloned in a vector for E. coli, and the fragment excisedfrom the vector was labeled using [³²P]-dCTP. This probe was contactedwith the nylon membrane prepared previously under stringent conditionsfor hybridization, thereby identifying a colony containing a fragment ofMre11 gene. This colony was isolated, and the scolony hybridization wasrepeated similarly to above to obtain a single clone containing theMre11 gene fragment. From this plasmid DNA, the Mre11 fragment wasexcised by restriction enzyme tdigestion and cloned into another vectorcompatible for E. coli, thus obtaining a plasmid pMRE11tr.

(2) Preparation of a DNA Encoding a Variant of Mre11 Protein

Using the DNA encoding the Mre11 protein obtained in (1) above, inaccordance with a method of Hashimoto-Gotoh et al. [Hashimoto-Gotoh, T.,et al.: Gene, 152:271-275 (1995)], DNAs encoding the following proteinswere prepared: a protein (also referred to as D16A type Mre11 protein)wherein the C-terminal domain of the Mre11 protein was deleted, aprotein (also referred as to DC49 type Mre11 protein) wherein theasparatic acid at 16th position from the N-terminal of the Mre11 wassubstituted by alanine, and a protein wherein the C-terminal domain ofthe Mre11 protein was deleted and the asparatic acid at 16th positionfrom an N-terminal of the Mre11 was substituted by alanine. That is, onenucleotide substitution from gat to gct of the codon at 16th position(47th nucleotide was changed from a to c)was carried out for D16Avariant, and further the 643rd and 644th codons were changed from gctagt to gcc tag to introduce a termination codon, for DC49 type variant.

(3) Construction of a Recombinant Vector Containing a DNA Encoding Mre11Protein or a Variant of Mre11 Protein

The DNA fragments obtained in (1) and (2) above which encode Mre11protein and a variant of Mre11 protein, as shown in FIG. 1, wereinserted at an EcoRI site present between ADH1 promoter and CYC1terminator in a yeast-E. coli shuttle vector including an aureobasidinresistant gene, thereby constructing a vector which enables high levelexpression in yeast. The recombinant vector containing a DNA encodingwild type Mre11 protein was named as pMACMre11WT, the recombinant vectorcontaining a DNA encoding Mre11 protein with the asparatic acid at 16thposition from the N-terminal substituted by alanine was aspMACMre11D16A, the recombinant vector containing a DNA encoding Mre11protein deleted a C-terminal domain was as pMACMre11ΔC49, and therecombinant vector including a DNA encoding Mre11 protein wherein theasparatic acid at the 16th position from N-terminal was substituted byalanine and C-terminal domain was deletedwas as pMACMre11D16AΔC49.

EXAMPLE 2

Transformation of Yeast with a Recombinant Vector

Transformation of yeast Saccharomyces cerevisiae ORD149 strain (adiploid having arg4rv/arg4bg and trp1-289/trp1-289 alleles), which wasgiven by Dr. Alain Nicolas (Institut Curie, France). with therecombinant vector obtained in Example 1 was conducted, as follows. Theyeast strain was washed with 1M sorbitol and mixed with the vector.After the DNA was introduced into the yeast cells by electroporation,the cells (the yeast strain having mutation in trp1 gene and the vectorhaving a selectable marker TRP1 gene) were allowed to grow on atryptophan-free minimal agar medium containing 1M sorbitol (, and thecolonies having the plasmid were selected. These yeast colonies werefurther grown in a medium containing 0.2 μg/ml of aureobasidin.

EXAMPLE 3

Analysis of Telomere Length

The telomere length of each transformant obtained in Example 2 wasanalyzed as follows. That is, a yeast cell including only pMAC561aurvector and transformant yeast cells which were obtained in Example 2,each having one of the recombinant vectors, pMACMre11WT, pMACMre11D16A,pMACMre11ΔC49, and pMACMre11D16AΔC49 were cultured for 12-16 hours in anutrient medium containing 0.2 μg/ml of aureobasidin, and the cells wereharvested. Next, in accordance with a conventional method, genomic DNAwas extracted from the cell, and after digestion by a restriction enzymeXhoI, the DNA fragments were separated by agarose gel electrophoresis.The DNA fragments on the agarose gel were transferred onto a nylonmembrane with vacuum under an alkaline condition, and the membrane wasanalyzed by Southern hybridization according to a method of Church etal. [Church, G. M. et. al.: Proc. Natl. Acad. Sci. U.S.A., 81:1991-1995(1984)]. As a probe, an oligonucleotide labeled with aradioactive isotope p³² at 5′-endwhich comprises 20 nucleotides of5′-gtgtgtgtgtgtgtgtgtgt-3′ (SEQ ID NO: 13) was used. The hybridizationwas performed for 20 hours at 50° C. After washing, the radioactivesignals on the membrane were visualized by autoradiography or with animaging plate manufactured by Fuji Photo Film Co., Ltd.

A chromosome of the obtained yeast has a structure as shown in FIG. 2. Aregion with which the probe can hybridize exists at some midpoints (Xtype) in addition to telomeres at the chromosome terminals (Y′ type). Incomparison with the yeast carrying only a vector not containing the DNAencoding Mre11 protein, when wild type Mre11 protein or the D16A typeMre11 protein with a mutation in the nuclease domain was highlyexpressed, the shortening of telomeres in the telomere regions of both Xand Y types was observed (FIG. 3) In particular, the yeast in which theD16A type Mre11 protein with a mutation in the nuclease domain washighly expressed, the shortening of telomeres was remarkable. Incontrast, with regard to a cell line in which the DC49 type Mre11protein deleted the C-terminal region was highly expressed, theelongation of telomeres was observed in all telomere regions of X and Ytypes. In view of the forgoing, it is shown that telomeres can beelongated by introducing into a cell a DNA encoding a protein having thedeletion of C-terminal domain of Mre11 protein, and the telomeres can beshortened by introducinga DNA encoding Mre11 protein.

INDUSTRIAL APPLICABILITY

According to the present invention, there are provided a method ofcontrolling telomere length, a control agent for controlling telomerelength, and a gene therapeutic agent for telomere length-associateddisease.

Sequence List Free Text

-   SEQ ID NO: 9: synthesis DNA-   SEQ ID NO: 10: synthesis DNA-   SEQ ID NO: 11: synthesis DNA-   SEQ ID NO: 12: synthesis DNA-   SEQ ID NO: 13: synthesis DNA

1. A method of controlling telomere length wherein the method comprisesmodifying physiological activity of endogenous Mre11 protein in aeukaryotic cell.
 2. The method of controlling telomere length accordingto claim 1, wherein the modification of physiological activity of theendogenous Mre11 protein is carried out by introducing into a cell a DNAencoding foreign Mre11 protein or a DNA encoding a protein wherein thenuclease domain or the C-terminal domain of the foreign Mre11 protein ismodified, in a state such that the DNA can be expressed.
 3. The methodof controlling telomere length according to claim 2, wherein the foreignMre11 protein is the following protein (a) or (b): (a) a proteincomprising an amino acid sequence represented by SEQ ID NO: 2 or 4; or(b) a protein comprising an amino acid sequence which is the amino acidsequence represented by SEQ ID NO: 2 or 4 from, in, or to which one ormore amino acids are deleted, substituted or added, and havingphysiological activity of Mre11 protein.
 4. The method of controllingtelomere length according to claim 2, wherein the DNA encoding theforeign Mre11 protein is the following DNA (c) or (d): (c) a DNAcomprising a nucleotide sequence represented by SEQ ID NO: 1 or 3; or(d) a DNA which can hybridize with the DNA of (c) under a stringentcondition and encodes a protein having physiological activity of Mre11protein.
 5. The method of controlling telomere length according to claim2, wherein the protein having the modified nuclease domain of theforeign Mre11 protein is the following protein (e) or (f): (e) a proteincomprising an amino acid sequence represented by SEQ ID NO: 6; or f) aprotein comprising an amino acid sequence which is the amino acidsequence represented by SEQ ID NO: 6 from, in, or to which one or moreamino acids are deleted, substituted or added, and having physiologicalactivity (except nuclease activity) of Mre11 protein.
 6. The method ofcontrolling telomere length according to claim 2, wherein the DNAencoding the protein having the modified nuclease domain of the foreignMre11 protein is the following DNA (g) or (h): (g) a DNA comprising anucleotide sequence represented SEQ ID NO: 5; or (h) a DNA which canhybridize with the DNA of (g) under a stringent condition and encodes aprotein having physiological activity (except nuclease activity) ofMre11 protein.
 7. The method of controlling telomere length according toclaim 2, wherein the protein having the modified C-terminal domain ofthe foreign Mre11 protein is the following protein (i) or (j): (i) aprotein comprising an amino acid sequence represented by SEQ ID NO: 8;or (j) a protein comprising an amino acid sequence which is the aminoacid sequence represented by SEQ ID NO: 8 from, in or to which one ormore amino acids are deleted, substituted or added, and havingphysiological activity (except double-stranded DNA binding activity) ofMre11 protein.
 8. The method of controlling telomere length according toclaim 2, wherein the DNA encoding the protein having the modifiedC-terminal domain of the foreign Mre11 protein is the following DNA (k)or (1): (k) a DNA comprising a nucleotide sequence represented by SEQ IDNO: 7; or (l) a DNA which can hybridize with the DNA of (k) and encodesa protein having physiological activity (except double-stranded DNAbinding activity) of Mre11 protein.
 9. An agent for controlling telomerelength comprising as an active component a substance which modifiesphysiological activity of endogenous Mre11 protein in a eukaryotic cell.10. The agent for controlling telomere length according to claim 9,wherein the substance is a DNA construct which comprises a DNA encodinga foreign Mre11 protein or a DNA encoding a protein wherein the nucleasedomain or the C-terminal domain of the Mre11 protein is modified in astate such that the DNA can be expressed.
 11. The agent for controllingtelomere length according to claim 10, wherein the foreign Mre11 proteinis the following protein (a) or (b): (a) a protein comprising an aminoacid sequence represented by SEQ ID NO: 2 or 4; or (b) a proteincomprising an amino acid sequence which is the amino acid sequencerepresented by SEQ ID NO: 2 or 4 from, in, or to which one or more aminoacids are deleted, substituted or added, and having physiologicalactivity of Mre11 protein.
 12. The agent for controlling telomere lengthaccording to claim 10, wherein the DNA encoding the foreign Mre11protein is the following DNA (c) or (d): (c) a DNA comprising anucleotide sequence represented by SEQ ID NO: 1 or 3; or (d) a DNA whichcan hybridize with the DNA of (c) under a stringent condition andencodes a protein having physiological activity of Mre11 protein. 13.The agent for controlling telomere length according to claim 10, whereinthe protein having the modified nuclease domain of the foreign Mre11protein is the following protein (e) or (f): (e) a protein comprising anamino acid sequence represented by SEQ ID NO: 6; or (f) a proteincomprising an amino acid sequence which is the amino acid sequencerepresented by SEQ ID NO: 6 from, in, or to. which one or more aminoacids are deleted, substituted or added, and having physiologicalactivity (except nuclease activity) of Mre11 protein.
 14. The agent forcontrolling telomere length according to claim 10, wherein the DNAencoding the protein having the modified nuclease domain of the foreignMre11 protein is the following DNA (g) or (h): (g) a DNA comprising anucleotide sequence represented SEQ ID NO: 5; or (h) a DNA which canhybridize with the DNA of (g) under a stringent condition and encodes aprotein having physiological activity (except nuclease activity) ofMre11 protein.
 15. The agent for controlling telomere length accordingto claim 10, wherein the protein having the modified C-terminal domainof the foreign Mre11 protein is the following protein (i) or (): (i) aprotein comprising an amino acid sequence represented by SEQ ID NO: 8;or (j) a protein comprising an amino acid sequence which is the aminoacid sequence represented by SEQ ID NO: 8 from, in or to which one ormore amino acids are deleted, substituted or added, and havingphysiological activity (except double-stranded DNA binding activity) ofMre11 protein.
 16. The agent for controlling telomere length accordingto claim 10, wherein the DNA encoding the protein having the modifiedC-terminal domain of the foreign Mre11 protein is the following DNA (k)or (1): (k) a DNA comprising a nucleotide sequence represented by SEQ IDNO: 7; or (l) a DNA which can hybridize with the DNA of (g) and encodesa protein having physiological activity (except double-stranded DNAbinding activity) of Mre11 protein.
 17. A gene therapeutic agent for atelomere length-associated disease comprising as an active agent asubstance which modifies physiological activity of endogenous Mre11protein in a eukaryotic cell.
 18. The gene therapeutic agent accordingto claim 17, wherein the substance is a DNA construct which comprises aDNA encoding foreign Mre11 protein or a DNA encoding a protein whereinthe nuclease domain or the C-terminal domain of the Mre11 protein ismodified, in a state such that the DNA can be expressed.
 19. The genetherapeutic agent according to claim 17, wherein the telomerelength-associated disease is a malignant tumor or a cell senescentdisease.
 20. The gene therapeutic agent according to claim 19, whereinthe malignant tumor is at least one selected from the group consistingof melanoma, hepatoma breast cancer, gastric cancer, and brain tumor.21. The gene therapeutic agent according to claim 18, wherein theforeign Mre11 protein is the following protein (a) or (b): (a) a proteincomprising an amino acid sequence represented by SEQ ID NO: 2 or 4; or(b) a protein comprising an amino acid sequence which is the amino acidsequence represented by SEQ ID NO: 2 or 4 from, in, or to which one ormore amino acids are deleted, substituted or added, and havingphysiological activity of Mre11 protein.
 22. The gene therapeutic agentaccording to claim 18, wherein the DNA encoding the foreign Mre11protein is the following DNA (c) or (d): (c) a DNA comprising anucleotide sequence represented by SEQ ID NO: 1 or 3; or (d) a DNA whichcan hybridize with the DNA of (c) under a stringent condition andencodes a protein having physiological activity of Mre11 protein. 23.The gene therapeutic agent according to claim 18, wherein the proteinhaving the modified nuclease domain of the foreign Mre11 protein is thefollowing protein (e) or (f): (e) a protein comprising an amino acidsequence represented by SEQ ID NO: 6; or (f) a protein comprising anamino acid sequence which is the amino acid sequence represented by SEQID NO: 6 from, in, or to which one or more amino acids are deleted,substituted or added, and having physiological activity (except nucleaseactivity) of Mre11 protein.
 24. The gene therapeutic agent according toclaim 18, wherein the DNA encoding the protein having the modifiednuclease domain of the foreign Mre11 protein is the following DNA (g) or(h): (g) a DNA comprising a nucleotide sequence represented SEQ ID NO:5; or (h) a DNA which can hybridize with the DNA of (g) under astringent condition and encodes a protein having physiological activity(except nuclease activity) of Mre11 protein.
 25. The gene therapeuticagent according to claim 18, wherein the protein having the modifiedC-terminal domain of the foreign Mre11 protein is the following protein(i) or (): (i) a protein comprising an amino acid sequence representedby SEQ ID NO: 8; or (j) a protein comprising an amino acid sequencewhich is the amino acid sequence represented by SEQ ID NO: 8 from, in orto which one or more amino acids are deleted, substituted or added, andhaving physiological activity (except double-stranded DNA bindingactivity) of Mre11 protein.
 26. The gene therapeutic agent according toclaim 18, wherein the DNA encoding the protein having the modifiedC-terminal domain of the foreign Mre11 protein is the following DNA (k)or (1): (k) a DNA comprising a nucleotide sequence represented by SEQ IDNO: 7; or (l) a DNA which can hybridize with the DNA of (g) and encodesa protein having physiological activity (except double-stranded DNAbinding activity) of Mre11 protein.